Trehalose Alleviates D-Galactose-Induced Aging-Related Granulosa Cell Death in Ovaries

Abstract

Ovarian dysfunction caused by aging restricts female reproductive capacity and is accompanied by oxidative stress and impaired autophagy. Recent studies have shown that trehalose (Tre) can activate autophagy and have antioxidant effects. However, whether Tre can be used to attenuate ovarian aging remains unclear. Therefore, the anti-aging effects of Tre on the ovary were explored both in vivo and in vitro. D-galactose (D-gal) was administered i.p. daily (200 mg/kg body weight) for 8 weeks to establish the mouse ovarian aging model (n = 10). We found that Tre significantly reversed ovarian weight loss and reduced the number of TUNEL-positive granulosa cells caused by D-gal in mouse ovaries. Tre elevated the protein expression levels of LC3-II, Parkin, PINK1, Beclin1, and LAMP2 in ovaries. Mitochondrial-related proteins TOM20 and COX IV expression levels were increased by Tre administration. In vitro studies further supported these findings, showing that Tre treatment significantly reduced the number of SA-β-gal and PI-positive cells, and decreased ROS levels in cultured granulosa cells. Thus, Tre alleviates ovarian aging by activating mitophagy and reducing oxidative stress, suggesting its potential as an anti-aging agent for ovarian health.

Keywords: aging; granulosa cell; mitophagy; ovary; trehalose.

Figure 1

Tre attenuates D-gal-induced apoptosis in granulosa cells. (A) Female mice were treated with D-gal (200 mg/kg body weight), Tre (2 g/kg body weight), or normal saline. (B) Histological changes in ovaries were examined by hematoxylin and eosin staining. Bar = 200 μm. (C) Quantification analysis of ovary weight/body weight. n = 10. (D) Quantification analysis of TUNEL-positive cells per follicles. (E) Representative images of TUNEL staining. Bar = 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl, control group. D-gal, D-gal group. Tre+D-gal, Tre+D-gal group.

Figure 2

Tre attenuates oxidative stress induced by D-gal in mouse ovaries. (A) Representative images of fluorescence-labeled Nrf2 and HO-1. Bar = 50 μm. (B,C) Fluorescence intensity quantification of Nrf2 and HO-1. (D–H) The expression levels of LC3, Parkin, PINK1, and TOM20 were detected by Western blot. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl, control group. D-gal, D-gal group. Tre+D-gal, Tre+D-gal group.

Figure 3

Tre activates mitophagy in D-gal-treated ovaries. (A) Representative images of fluorescence-labeled Parkin and PINK1. Bar = 50 μm. (B,C) Fluorescence intensity quantification of Parkin and PINK1. (D) Representative images of fluorescence-labeled LC3 and COX IV. Bar = 50 μm. (E,F) Fluorescence intensity quantification of LC3 and COX IV. * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl, control group. D-gal, D-gal group. Tre+D-gal, Tre+D-gal group.

Figure 4

Tre promotes autophagic flux in granulosa cells of ovaries. Immunofluorescence of p62 and Beclin1 in Ctrl, D-gal, Tre+D-gal groups. Bar = 50 μm. * p < 0.05, *** p < 0.001. Ctrl, control group. D-gal, D-gal group. Tre+D-gal, Tre+D-gal group.

Figure 5

Tre induces lysosomal biogenesis in granulosa cells of ovaries. Immunofluorescence of LAMP2 and PCNA in Ctrl, D-gal, Tre+D-gal groups. Bar = 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl, control group. D-gal, D-gal group. Tre+D-gal, Tre+D-gal group.

Figure 6

Tre alleviates D-gal-induced granulosa cell death in vitro. (A) Granulosa cells were isolated and stimulated with different concentrations of D-gal (15, 30, 45, and 60 mg/mL) for 24 h. CCK-8 assay was performed to detect the granulosa cell viability. n = 5. (B,C) Granulosa cells were pretreated with 10 mmol/L Tre for 24 h, then exposed to 45 mg/mL D-gal for 24 h. Cell senescence was examined using SA-β-gal staining. n = 5. Bar = 100 μm. (D) Representative images of fluorescence-labeled PI. Bar = 100 μm. (E) Quantification analysis of PI-positive cells. n = 6. (F,G) ROS levels were determined by DCFH-DA staining in different groups. n = 5. Bar = 100 μm. ns, no significance. * p < 0.05, *** p < 0.001. Ctrl, control group. D-gal, D-gal group. Tre+D-gal, Tre+D-gal group.

Figure 7

Schematic illustration of Tre attenuates D-gal-induced ovarian aging in mice.